PD-1 Antibodies and PD-L1 Antibodies and Uses Thereof

ABSTRACT

The invention relates to PD-1 antibodies and PD-L1 antibodies and uses thereof.

FIELD OF THE INVENTION

The invention relates to PD-1 antibodies and PD-L1 antibodies and usesthereof.

BACKGROUND OF THE INVENTION

Co-receptor signalling is an important mechanism for coordinating andtightly regulating immune responses. The usual scheme of activation ofαβ T cells relies on positive signals given by peptide antigenspresented by HLA class I or II. Co-receptor signals will either increaseor prevent this activation.

Among the negative signalling molecules, those belonging to CD28/B7families are by far the most studied. Three members of this family havebeen described: CTL-associated antigen-4 (CTLA-4), programmed death-1(PD-1) and B and T lymphocyte attenuator (BTLA). They all play a role inthe control of tolerance. They provide negative signals that limit,terminate and/or attenuate immune responses.

PD-1 was isolated as a gene up-regulated in a T cell hybridomaundergoing apoptosis and was named program death 1. PD-1 or CD279 isexpressed on activated T and B cells as well as on activated myeloidcells.

Its expression is broader than CTLA-4 which is only found on activated Tcells.

Upon coligation with the T cell Receptor (TcR), PD-1 elicits inhibitorysignals.

The PD-1 cytoplasmic domain contains two tyrosines, one that constitutesan immunoreceptor tyrosine inhibitory receptor (ITIM) and the other onean immunoreceptor tyrosine based switch motif (ITSM). Thephosphorylation of the second tyrosine leads to the recruitment of thetyrosine phosphatases SHP2 and to some extent SHP1. These phosphataseswill dephosphorylate ZAP70, CD3ξ and PKCθ and consequently willattenuate T cell signals.

PD-1 mainly inhibits T and B cell proliferation by causing cell arrestin G0/G1 and inhibiting cytokine production in T cells.

Two PD-1 ligands have been described, PD-L1/B7H1/CD274 andPD-L2/B7-DC/CD273. PD-L1 is expressed at low levels on immune cells suchas B cells, dendritic cells, macrophages and T cells and is up regulatedfollowing activation. PD-L1 is also expressed on non-lymphoid organssuch as endothelial cells, heart, lung, pancreas, muscle, keratinocytesand placenta. The expression within non lymphoid tissues suggests thatPD-L1 may regulate the function of self reactive T and B cells as wellas myeloid cells in peripheral tissues or may regulate inflammatoryresponses in the target organs. PD-L1 expression is mainly regulated bytype 1 and 2 interferon which are major regulators of PD-L1 onendothelial and epithelial cells. PD-L1 is expressed in tumor samplesand is associated to poor prognosis. Various viral infections induce theintense PD-L1 expression on host tissues.

PD-L2/B7-DC cell surface expression is restricted to macrophages anddendritic cells, though PD-L2 transcript was found in non hematopietictissues such as heart, liver and pancreas. Its surface expressiondepends on the production of IFNγ and Th2 cytokines.

PD-L1 and PD-L2 expression depends also on distinct stimuli. Onmacrophages PD-L1 is induced by INFγ whereas PD-L2 is induced by IL-4. Asimilar regulation is found on DC though these differences are notabsolute. These studies tend to suggest that PD-L1 might regulatepreferentially Th1 responses whereas PD-L2 would regulate Th2 responses.

Both PD-L1 and PD-L2 inhibit T cell proliferation, cytokine productionand β1 and β2 integrins mediated adhesion. Although some contradictorydata have proposed a costimulatory function. However, PD-L2 but notPD-L1 triggers reverse signalling in dendritic cells leading to IL-12production and activation of T cells.

The expression patterns of PD-L1 and PD-L2 suggest both overlapping anddifferential roles in immune regulation. PD-L1 is abundant in a varietyof human cancers (Dong et al (2002) Nat. Med 8:787-9). The interactionbetween PD-1 and PD-L1 results in a decrease in tumor infiltratinglymphocytes, a decrease in T-cell receptor mediated proliferation, andimmune evasion by the cancerous cells (Dong et al. (2003) J. Mol. Med.81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314;Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppressioncan be reversed by inhibiting the local interaction of PD-1 with PD-L1,and the effect is additive when the interaction of PD-1 with PD-L2 isblocked as well (Iwai et al. (2002) Proc. Nat 7. Acad. Sci USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

PD-1 deficient animals develop various autoimmune phenotypes, includingautoimmune cardiomyopathy and a lupus-like syndrome with arthritis andnephritis (Nishimura et al. (1999) Immunity H: 141-51; Nishimura et al.(2001) Science 291:319-22). Additionally, PD-1 has been found to play arole in autoimmune encephalomyelitis, systemic lupus erythematosus,graft-versus-host disease (GVHD), type I diabetes, and rheumatoidarthritis (Salama et al. (2003) J Exp Med 198:71-78: Prokunina andAlarcon-Riquelme (2004) Hum MoI Genet 13:R143; Nielsen et al. (2004)Lupus 11:510).

In animal models, PD-L1 and PD-L2 blockade using blocking mAbs evidencedistinct roles in the susceptibility and chronic progression ofexperimental autoimmune encephalitis in a strain specific manner. In NODprediabetic mice PD-L1 but not PD-L2 blockade precipitated diabetes.Using the RIP-mOVA mouse model of autoimmune diabetes, Martin-Orozco etal. found that PD-L1 but not PD-L2 mediated the inhibition of diabetesonset (Martin-Orozco et al. (2006) J Immunol. 15; 177(12):8291-5).

To date, no satisfactory approach has been proven to induce potentimmune responses against vaccines, especially in cancer patients.Methods have yet to be devised to overcome the immunosuppressivemechanisms observed in cancer patients, and during chronic infections.

Treatment of autoimmune diseases and prevention of transplantationrejection in graft versus host diseases (GVHD) depends onimmunosuppressive agents that have serious side effects, or are notalways effective. New immunosuppressive agents are desired.

SUMMARY OF THE INVENTION

The present invention relates to a PD-1 antibody (PD1.3) which isobtainable from the hybridoma accessible under CNCM deposit numberI-4122.

The invention also relates to a PD-1 antibody which comprises the CDRsof PD1.3.

The invention relates to PD1.3 or a derivative thereof for the use in amethod for treatment of the human or animal body by therapy.

The invention relates to PD1.3 or a derivative thereof for the treatmentof a cancer or a chronic infection.

The invention relates to a vaccine for the treatment of a cancer or achronic infection comprising PD1.3 or a derivative thereof.

The invention relates to a kit for the treatment of a cancer or achronic infection comprising:

-   -   a) PD1.3 or a derivative thereof; and    -   b) a vaccine for the treatment of a cancer or a chronic        infection.

The present invention also relates to a PD-L1 antibody, which stabilizesthe binding of PD-L1 to PD-1.

The invention relates to a PD-L1 antibody, which stabilizes the bindingof PD-L1 to PD-1 for the use in a method for treatment of the human oranimal body by therapy.

The invention relates to a PD-L1 antibody, which stabilizes the bindingof PD-L1 to PD-1 for the treatment of an autoimmune disease,transplantation rejection or a graft versus host disease.

DETAILED DESCRIPTION OF THE INVENTION Definitions

According to the present invention, “antibody” or “immunoglobulin” havethe same meaning, and will be used equally in the present invention. Theterm “antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody fragments or derivatives. Antibodyfragments include but are not limited to Fv, Fab, F(ab′)₂, Fab′, dsFv,scFv, sc(Fv)₂ and diabodies.

In natural antibodies, two heavy chains are linked to each other bydisulfide bonds and each heavy chain is linked to a light chain by adisulfide bond. There are two types of light chain, lambda (λ) and kappa(κ). There are five main heavy chain classes (or isotypes) whichdetermine the functional activity of an antibody molecule: IgM, IgD,IgG, IgA and IgE. Each chain contains distinct sequence domains. Thelight chain includes two domains, a variable domain (VL) and a constantdomain (CL). The heavy chain includes four domains, a variable domain(VH) and three constant domains (CH1, CH2 and CH3, collectively referredto as CH). The variable regions of both light (VL) and heavy (VH) chainsdetermine binding recognition and specificity to the antigen. Theconstant region domains of the light (CL) and heavy (CH) chains conferimportant biological properties such as antibody chain association,secretion, trans-placental mobility, complement binding, and binding toFc receptors (FcR). The Fv fragment is the N-terminal part of the Fabfragment of an immunoglobulin and consists of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site. Complementarity Determining Regions or CDRsrefer to amino acid sequences which together define the binding affinityand specificity of the natural Fv region of a native immunoglobulinbinding site. The light and heavy chains of an immunoglobulin each havethree CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2,H-CDR3, respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. Framework Regions (FRs) refer to amino acid sequences interposedbetween CDRs.

The terms “chimeric antibody” refer to a genetically engineered fusionof parts of an animal antibody, typically a mouse antibody, with partsof a human antibody. Generally, chimeric antibodies containapproximately 33% mouse protein and 67% human protein. Developed toreduce the Human Anti-animal Antibodies response elicited by animalantibodies, they combine the specificity of the animal antibody with theefficient human immune system interaction of a human antibody.

According to the invention, the term “humanized antibody” refers to anantibody having variable region framework and constant regions from ahuman antibody but retains the CDRs of the animal antibody.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)₂” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

The term “Fab′” refers to an antibody fragment having a molecular weightof about 50,000 and antigen binding activity, which is obtained bycutting a disulfide bond of the hinge region of the F(ab′)₂.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. “dsFv” is aVH::VL heterodimer stabilised by a disulfide bond. Divalent andmultivalent antibody fragments can form either spontaneously byassociation of monovalent scFvs, or can be generated by couplingmonovalent scFvs by a peptide linker, such as divalent sc(Fv)₂.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

By “purified” and “isolated” it is meant, when referring to apolypeptide (i.e. an antibody according to the invention) or to anucleotide sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type are present. An “isolated”nucleic acid molecule which encodes a particular polypeptide refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the polypeptide; however, the molecule mayinclude some additional bases or moieties which do not deleteriouslyaffect the basic characteristics of the composition.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition. A “therapeuticallyeffective amount” is intended for a minimal amount of active agent whichis necessary to impart therapeutic benefit to a subject. For example, a“therapeutically effective amount” to a mammal is such an amount whichinduces, ameliorates or otherwise causes an improvement in thepathological symptoms, disease progression or physiological conditionsassociated with or resistance to succumbing to a disorder.

As used herein, the term “prevention” refers to preventing the diseaseor condition from occurring in a subject which has not yet beendiagnosed as having it.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a subject according to theinvention is a human.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. The terms “cancer” or “neoplasms” include malignancies ofthe various organ systems, such as affecting lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The inventors have deposited a murine PD-1 antibody (PD1.3) producinghybridoma at the Collection Nationale de Cultures de Microorganismes(CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15,France), in accordance with the terms of Budapest Treaty, on Feb. 4,2009. The deposited hybridoma has CNCM deposit number I-4122.

“PD1.3” refers to an isolated PD-1 antibody which is obtainable from thehybridoma accessible under CNCM deposit number I-4122

The expression “a derivative of PD1.3” refers to a PD-1 antibody whichcomprises the 6 CDRs of PD1.3.

The inventors have deposited a murine PD-L1 antibody (PDL1.1) producinghybridoma at the Collection Nationale de Cultures de Microorganismes(CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15,France), in accordance with the terms of Budapest Treaty, on Oct. 15,2008. The deposited hybridoma has CNCM deposit number I-4080.

“PDL1.1” refers to an isolated PD-L1 antibody which is obtainable fromthe hybridoma accessible under CNCM deposit number I-4080.

The expression “a derivative of PDL1.1” refers to a PD-L1 antibody whichcomprises the 6 CDRs of PDL1.1.

The inventors have deposited a murine PD-L1 antibody (PDL1.2) producinghybridoma at the Collection Nationale de Cultures de Microorganismes(CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15,France), in accordance with the terms of Budapest Treaty, on Oct. 15,2008. The deposited hybridoma has CNCM deposit number I-4081.

“PDL1.2” refers to an isolated PD-L1 antibody which is obtainable fromthe hybridoma accessible under CNCM deposit number I-4081.

The expression “a derivative of PDL1.2” refers to a PD-L1 antibody whichcomprises the 6 CDRs of PDL1.2.

Antibodies of the Invention and Nucleic Acids Encoding them

The present invention relates to an isolated PD-1 antibody (PD1.3) whichis obtainable from the hybridoma accessible under CNCM deposit numberI-4122.

The present invention relates to the hybridoma accessible under CNCMdeposit number I-4122.

The invention relates to an antibody which comprises the 6 CDRs ofPD1.3.

In another embodiment, the invention relates to a derivative of PD1.3which comprises the VL chain and the VH chain of PD1.3.

In another embodiment, the invention relates to a derivative of PD1.3which is a chimeric antibody, which comprises the variable domains ofPD1.3.

The present invention also relates to an isolated PD-L1 antibody, whichstabilizes the binding of PD-L1 to PD-1.

Typically the stabilization of binding of PD-L1 to PD-1 may be measuredaccording to the method described in the example.

Examples of isolated PD-L1 antibodies, which stabilize the binding ofPD-L1 to PD-1 are PDL1.1, PDL1.2 or derivatives thereof.

The present invention also relates to the hybridomas accessible underCNCM deposit number I-4080 or I-4081.

The invention also relates to an antibody which comprises the 6 CDRs ofPDL1.1 or the 6 CDRs of PDL1.2.

In another embodiment, the invention relates to a derivative of PDL1.1or PDL1.2 which comprises the VL chain and the VH chain of PDL1.1 orPDL1.2 respectively.

In another embodiment, the invention relates to a derivative of PDL1.1or PDL1.2 which is a chimeric antibody, which comprises the variabledomains of PDL1.1 or PDL1.2.

In an embodiment, an antibody of the invention is a monoclonal antibody.

In an embodiment, an antibody of the invention is a chimeric antibody.

In an embodiment, an antibody of the invention is a humanized antibody.

A further embodiment of the invention relates to a nucleic acid sequenceencoding an antibody of the invention.

In a particular embodiment, the invention relates to a nucleic acidsequence encoding the VH domain or the VL domain of an antibody of theinvention.

Typically, said nucleic acid is a DNA or RNA molecule, which may beincluded in any suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence.

So, a further object of the invention relates to a vector comprising anucleic acid of the invention.

Such vectors may comprise regulatory elements, such as a promoter,enhancer, terminator and the like, to cause or direct expression of saidantibody upon administration to a subject. Examples of promoters andenhancers used in the expression vector for animal cell include earlypromoter and enhancer of SV40, LTR promoter and enhancer of Moloneymouse leukemia virus, promoter and enhancer of immunoglobulin H chainand the like.

Any expression vector for animal cell can be used, so long as a geneencoding the human antibody C region can be inserted and expressed.Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR,pSG1 beta d2-4- and the like.

Other examples of plasmids include replicating plasmids comprising anorigin of replication, or integrative plasmids, such as for instancepUC, pcDNA, pBR, and the like.

Other examples of viral vector include adenoviral, retroviral, herpesvirus and AAV vectors. Such recombinant viruses may be produced bytechniques known in the art, such as by transfecting packaging cells orby transient transfection with helper plasmids or viruses. Typicalexamples of virus packaging cells include PA317 cells, PsiCRIP cells,GPenv+ cells, 293 cells, etc. Detailed protocols for producing suchreplication-defective recombinant viruses may be found for instance inWO 95/14785, WO 96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478.

A further object of the present invention relates to a cell which hasbeen transfected, infected or transformed by a nucleic acid and/or avector according to the invention. The term “transformation” means theintroduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNAor RNA sequence to a host cell, so that the host cell will express theintroduced gene or sequence to produce a desired substance, typically aprotein or enzyme coded by the introduced gene or sequence. A host cellthat receives and expresses introduced DNA or RNA has been“transformed”.

The nucleic acids of the invention may be used to produce an antibody ofthe invention in a suitable expression system. The term “expressionsystem” means a host cell and compatible vector under suitableconditions, e.g. for the expression of a protein coded for by foreignDNA carried by the vector and introduced to the host cell.

Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors. Other examples of host cells include, withoutlimitation, prokaryotic cells (such as bacteria) and eukaryotic cells(such as yeast cells, mammalian cells, insect cells, plant cells, etc.).Specific examples include E. coli, Kluyveromyces or Saccharomycesyeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells,COS cells, etc.) as well as primary or established mammalian cellcultures (e.g., produced from lymphoblasts, fibroblasts, embryoniccells, epithelial cells, nervous cells, adipocytes, etc.). Examples alsoinclude mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell(ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene(hereinafter referred to as “DHFR gene”) is defective, ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”), and the like.

The present invention also relates to a method of producing arecombinant host cell expressing an antibody according to the invention,said method comprising the steps of: (i) introducing in vitro or ex vivoa recombinant nucleic acid or a vector as described above into acompetent host cell, (ii) culturing in vitro or ex vivo the recombinanthost cell obtained and (iii), optionally, selecting the cells whichexpress and/or secrete said antibody. Such recombinant host cells can beused for the production of antibodies of the invention.

Methods of Producing Antibodies of the Invention

Antibodies of the invention may be produced by any technique known inthe art, such as, without limitation, any chemical, biological, geneticor enzymatic technique, either alone or in combination.

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said antibodies, by standard techniques forproduction of polypeptides. For instance, they can be synthesized usingwell-known solid phase method, preferably using a commercially availablepeptide synthesis apparatus (such as that made by Applied Biosystems,Foster City, Calif.) and following the manufacturer's instructions.Alternatively, antibodies of the invention can be synthesized byrecombinant DNA techniques well-known in the art. For example,antibodies can be obtained as DNA expression products afterincorporation of DNA sequences encoding the antibodies into expressionvectors and introduction of such vectors into suitable eukaryotic orprokaryotic hosts that will express the desired antibodies, from whichthey can be later isolated using well-known techniques.

In particular, the invention further relates to a method of producing anantibody of the invention, which method comprises the steps consistingof: (i) culturing a transformed host cell according to the inventionunder conditions suitable to allow expression of said antibody; and (ii)recovering the expressed antibody.

In another particular embodiment, the method comprises the steps of:

(i) culturing the hybridoma deposited as CNCM I-4122, CNCM I-4080 orCNCM I-4081 under conditions suitable to allow expression of theantibody; and(ii) recovering the expressed antibody.

Antibodies of the invention are suitably separated from the culturemedium by conventional immunoglobulin purification procedures such as,for example, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In a particular embodiment, the human chimeric antibody of the presentinvention can be produced by obtaining nucleic sequences encoding VL andVH domains as previously described, constructing a human chimericantibody expression vector by inserting them into an expression vectorfor animal cell having genes encoding human antibody CH and humanantibody CL, and expressing the coding sequence by introducing theexpression vector into an animal cell.

As the CH domain of a human chimeric antibody, it may be any regionwhich belongs to human immunoglobulin, but those of IgG class aresuitable and any one of subclasses belonging to IgG class, such as IgG1,IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a humanchimeric antibody, it may be any region which belongs to Ig, and thoseof kappa class or lambda class can be used.

Methods for producing chimeric antibodies involve conventionalrecombinant DNA and gene transfection techniques are well known in theart (See patent documents U.S. Pat. No. 5,202,238; and U.S. Pat. No.5,204,244).

The humanized antibody of the present invention may be produced byobtaining nucleic acid sequences encoding CDR domains, as previouslydescribed, constructing a humanized antibody expression vector byinserting them into an expression vector for animal cell having genesencoding (i) a heavy chain constant region identical to that of a humanantibody and (ii) a light chain constant region identical to that of ahuman antibody, and expressing the genes by introducing the expressionvector into an animal cell.

The humanized antibody expression vector may be either of a type inwhich a gene encoding an antibody heavy chain and a gene encoding anantibody light chain exists on separate vectors or of a type in whichboth genes exist on the same vector (tandem type). In respect ofeasiness of construction of a humanized antibody expression vector,easiness of introduction into animal cells, and balance between theexpression levels of antibody H and L chains in animal cells, humanizedantibody expression vector of the tandem type is preferred. Examples oftandem type humanized antibody expression vector include pKANTEX93 (WO97/10354), pEE18 and the like.

Methods for producing humanized antibodies based on conventionalrecombinant DNA and gene transfection techniques are well known in theart. Antibodies can be humanized using a variety of techniques known inthe art including, for example, CDR-grafting (EP 239,400; PCTpublication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596), and chainshuffling (U.S. Pat. No. 5,565,332). The general recombinant DNAtechnology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and International PatentApplication WO 96/02576). The Fab of the present invention can beobtained by treating an antibody which specifically reacts with PD-1with a protease, papaine. Also, the Fab can be produced by inserting DNAencoding Fab of the antibody into a vector for prokaryotic expressionsystem, or for eukaryotic expression system, and introducing the vectorinto a procaryote or eucaryote (as appropriate) to express the Fab.

The F(ab′)₂ of the present invention can be obtained treating anantibody which specifically reacts with PD1.3 with a protease, pepsin.Also, the F(ab′)₂ can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

The Fab′ of the present invention can be obtained treating F(ab′)₂ whichspecifically reacts with human PD-1 with a reducing agent,dithiothreitol. Also, the Fab′ can be produced by inserting DNA encodingFab′ fragment of the antibody into an expression vector for prokaryote,or an expression vector for eukaryote, and introducing the vector into aprokaryote or eukaryote (as appropriate) to perform its expression.

The scFv of the present invention can be produced by obtaining cDNAencoding the VH and VL domains as previously described, constructing DNAencoding scFv, inserting the DNA into an expression vector forprokaryote, or an expression vector for eukaryote, and then introducingthe expression vector into a prokaryote or eukaryote (as appropriate) toexpress the scFv. To generate a humanized scFv fragment, a well knowntechnology called CDR grafting may be used, which involves selecting thecomplementary determining regions (CDRs) from a donor scFv fragment, andgrafting them onto a human scFv fragment framework of known threedimensional structure (see, e.g., W098/45322; WO 87/02671; U.S. Pat. No.5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567; EP0173494).

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody. Itis known that when a humanized antibody is produced by simply graftingonly CDRs in VH and VL of an antibody derived from a non-human animal inFRs of the VH and VL of a human antibody, the antigen binding activityis reduced in comparison with that of the original antibody derived froma non-human animal. It is considered that several amino acid residues ofthe VH and VL of the non-human antibody, not only in CDRs but also inFRs, are directly or indirectly associated with the antigen bindingactivity. Hence, substitution of these amino acid residues withdifferent amino acid residues derived from FRs of the VH and VL of thehuman antibody would reduce of the binding activity. In order to resolvethe problem, in antibodies grafted with human CDR, attempts have to bemade to identify, among amino acid sequences of the FR of the VH and VLof human antibodies, an amino acid residue which is directly associatedwith binding to the antibody, or which interacts with an amino acidresidue of CDR, or which maintains the three-dimensional structure ofthe antibody and which is directly associated with binding to theantigen. The reduced antigen binding activity could be increased byreplacing the identified amino acids with amino acid residues of theoriginal antibody derived from a non-human animal.

Modifications and changes may be made in the structure of the antibodiesof the present invention, and in the DNA sequences encoding them, andstill obtain a functional molecule that encodes an antibody withdesirable characteristics.

In making the changes in the amino sequences, the hydropathic index ofamino acids may be considered. The importance of the hydropathic aminoacid index in conferring interactive biologic function on a protein isgenerally understood in the art. It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophane (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate(−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

A further embodiment of the present invention also encompassesfunction-conservative variants of the antibodies of the presentinvention.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids otherthan those indicated as conserved may differ in a protein so that thepercent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide which has atleast 60% amino acid identity as determined by BLAST or FASTAalgorithms, preferably at least 75%, more preferably at least 85%, stillpreferably at least 90%, and even more preferably at least 95%, andwhich has the same or substantially similar properties or functions asthe native or parent protein to which it is compared.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than 80%, preferably greater than85%, preferably greater than 90% of the amino acids are identical, orgreater than about 90%, preferably greater than 95%, are similar(functionally identical) over the whole length of the shorter sequence.Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program, orany of sequence comparison algorithms such as BLAST, FASTA, etc.

For example, certain amino acids may be substituted by other amino acidsin a protein structure without appreciable loss of activity. Since theinteractive capacity and nature of a protein define the protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and, of course, in its DNA encodingsequence, while nevertheless obtaining a protein with like properties.It is thus contemplated that various changes may be made in theantibodies sequences of the invention, or corresponding DNA sequenceswhich encode said antibodies, without appreciable loss of theirbiological activity.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Another type of amino acid modification of theantibody of the invention may be useful for altering the originalglycosylation pattern of the antibody.

By “altering” is meant deleting one or more carbohydrate moieties foundin the antibody, and/or adding one or more glycosylation sites that arenot present in the antibody.

Glycosylation of antibodies is typically N-linked. “N-linked” refers tothe attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagines-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. Addition of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites).

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. For example, suchmethods are described in WO87/05330.

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Enzymatic cleavage of carbohydrate moieties onantibodies can be achieved by the use of a variety of endo- andexo-glycosidases.

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of non proteinaceous polymers, eg.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

It may be also desirable to modify the antibody of the invention withrespect to effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing inter-chain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and/or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. J Exp Med. 1992 Oct. 1; 176(4):1191-5 and Shopes B. J Immunol.1992 May 1; 148(9):2918-22).

Therapeutic Uses of the Antibodies of the Invention

The inventors have demonstrated that PD1.3 inhibits the binding of PD-L1and PD-L2 to PD-1 and thereby may be used to overcome theimmunosuppressive mechanisms mediated by PD-1 observed in cancerpatients and during chronic infections.

The invention relates to PD1.3 or a derivative thereof for the use in amethod for treatment of the human or animal body by therapy.

The invention relates to PD1.3 or a derivative thereof for the treatmentof a cancer or a chronic infection.

The invention also relates to a method for treating a cancer or achronic infection wherein said method comprises the step ofadministering to a subject in need thereof a therapeutically effectiveamount of PD1.3 or of a derivative thereof.

Examples of cancers include, but are not limited to, hematologicalmalignancies such as B-cell lymphoid neoplasm, T-cell lymphoid neoplasm,non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL),NK-cell lymphoid neoplasm and myeloid cell lineage neoplasm. Examples ofnon-hematological cancers include, but are not limited to, colon cancer,breast cancer, lung cancer, brain cancer, prostate cancer, head and neckcancer, pancreatic cancer, bladder cancer, colorectal cancer, bonecancer, cervical cancer, liver cancer, oral cancer, esophageal cancer,thyroid cancer, kidney cancer, stomach cancer, testicular cancer andskin cancer.

Examples of chronic infections include, but are not limited to, viral,bacterial, parasitic or fungal infections such as chronic hepatitis,lung infections, lower respiratory tract infections, bronchitis,influenza, pneumoniae and sexually transmitted diseases. Examples ofviral infections include, but are not limited to, hepatitis (HAV, HBV,HCV), herpes simplex (HSV), herpes zoster, HPV, influenza (Flu), AIDSand AIDS related complex, chickenpox (varicella), common cold,cytomegalovirus (CMV) infection, smallpox (variola), colorado tickfever, dengue fever, ebola hemorrhagic fever, foot and mouth disease,lassa fever, measles, marburg hemorrhagic fever, infectiousmononucleosis, mumps, norovirus, poliomyelitis, progressive multifocalleukencephalopathy (PML), rabies, rubella, SARS, viral encephalitis,viral gastroenteritis, viral meningitis, viral pneumonia, West Niledisease and yellow fever. Examples of bacterial infections include, butare not limited to, pneumonia, bacterial meningitis, cholera,diphtheria, tuberculosis, anthrax, botulism, brucellosis,campylobacteriosis, typhus, gonorrhea, listeriosis, lyme disease,rheumatic fever, pertussis (Whooping Cough), plague, salmonellosis,scarlet fever, shigellosis, syphilis, tetanus, trachoma, tularemia,typhoid fever, and urinary tract infections. Examples of parasiticinfections include include, but are not limited to, malaria,leishmaniasis, trypanosomiasis, chagas disease, cryptosporidiosis,fascioliasis, filariasis, amebic infections, giardiasis, pinworminfection, schistosomiasis, taeniasis, toxoplasmosis, trichinellosis,and trypanosomiasis. Examples of fungal infections include, but are notlimited to, candidiasis, aspergillosis, coccidioidomycosis,cryptococcosis, histoplasmosis and tinea pedis.

PD1.3 or a derivative thereof may be used as a vaccine adjuvant for thetreatment of a cancer or a chronic infection.

The invention relates to a vaccine for the treatment of a cancer or achronic infection comprising PD1.3 or a derivative thereof.

The invention relates to a kit for the treatment of a cancer or achronic infection comprising:

-   -   a) PD1.3 or a derivative thereof; and    -   b) a vaccine for the treatment of a cancer or a chronic        infection.

The two elements of the kit may be administered concomitantly orsequentially over time.

Examples of vaccine for the treatment of a cancer or a chronic infectionare:

include, but are not limited to vaccines against viral, bacterial,parasitic or fungal infections such as HIV and HBV and vaccines againstviral associated cancers (for instance HPV or HBV) or anti cancervaccines for instance used to treat patients with melanoma, leukemia,breast cancers, lung cancers.

Furthermore, the inventors have generated PD-L1 antibodies, whichstabilize the binding of PD-L1 to PD-1 and thereby may be used tostimulate the immunosuppressive mechanisms mediated by PD-1. These PD-L1antibodies may be used as immunosuppressive agents.

In a further embodiment, the invention relates to a PD-L1 antibody,which stabilizes the binding of PD-L1 to PD-1 for the use in a methodfor treatment of the human or animal body by therapy.

In particular, the invention relates to a PD-L1 antibody, whichstabilizes the binding of PD-L1 to PD-1 for the treatment of anautoimmune disease, transplantation rejection or a graft versus hostdisease.

The invention also relates to a method for treating an autoimmunedisease, transplantation rejection or a graft versus host disease,wherein said method comprises the step of administering to a subject inneed thereof a therapeutically effective amount of PD-L1 antibody, whichstabilizes the binding of PD-L1 to PD-1. Typically the PD-L1 antibodies,which stabilize the binding of PD-L1 to PD-1 may be PDL1.1, a derivativethereof, PDL1.2 or a derivative thereof.

Examples of autoimmune diseases which may be treated include but are notlimited to rheumatoid arthritis (RA), insulin dependent diabetesmellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn's disease,systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome,pemphigus vulgaris, pemphigoid, addison's disease, ankylosingspondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmunehepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome,Graves' disease, Guillain-Barré syndrome, Hashimoto's disease,idiopathic leucopenia, idiopathic thrombocytopenic purpura, maleinfertility, mixed connective tissue disease, myasthenia gravis,pernicious anemia, phacogenic uveitis, primary biliary cirrhosis,primary myxoedema, Reiter's syndrome, stiff man syndrome,thyrotoxicosis, ulceritive colitis, and Wegener's granulomatosis.

Typically a PD-L1 antibody, which stabilizes the binding of PD-L1 toPD-1 may be used in combination with other immunosuppressive andchemotherapeutic agents such as, but not limited to, prednisone,azathioprine, cyclosporin, methotrexate, and cyclophosphamide .

The invention also relates to pharmaceutical composition comprising anantibody of the invention.

Therefore, an antibody of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semI-3889solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment.

To prepare pharmaceutical compositions, an effective amount of theantibody may be dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

An antibody of the invention can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The antibodies of the invention may be formulated within a therapeuticmixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per doseor so. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; time release capsules ; and any other form currentlyused.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of antibodies into host cells. Theformation and use of liposomes and/or nanoparticles are known to thoseof skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) are generally designedusing polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 A, containing an aqueous solution in the core. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations.

The invention will further be illustrated in view of the followingfigures and example.

FIGURES

FIG. 1: SPR analysis using Biacore of PD-L1 and PD-L2 competitivebinding to PD-1.

(A) A schematic representation of the surface competitive bindinginhibition used in (B).

(B) The PD-1 chips were pre-incubated with increasing amount of PD-L2(from 0 to 1000 RU) and PD-L1 were injected at 10 μg/ml for 2 minutes ata flow rate of 10 μl/min without removing bound PD-L2. Sensorgramsshowing the PD-L1 binding at different level of PD-L2 occupancy aresuperimposed.

(C) A schematic representation of the solution inhibition used in (D).

(D) PD-1 recombinant proteins at 10 μg/ml were pre-incubated withincreasing concentrations of PD-L2 (from 0 to 60 μg/ml) and injected for2 minutes at a flow rate of 10 μl/min onto the PD-L1 chip. RU valuesmonitored 10 seconds after the end of injection were plotted as afunction of PD-L2 concentration (log scale).

FIG. 2: The PD1.3 antibody blocks the binding of PD-1 to both PD-L1 andPD-L2 and enhance T cell activation.

(A) A schematic representation of the surface competitive bindinginhibition used in (B) and (C). In a first step the immobilized PD-1proteins are saturated using the antibody Fabs and the correspondingPD-1 ligands are injected as a soluble analyte in a second step.

(B and C) PD-L1 Ig (B) and PD-L2 Ig proteins (C) were injected at 10μg/ml for two minutes at a flow rate of 10 μl/min onto PD-1 chip (none),or PD-1 chip pre-incubated with anti PD-1 Fab, PD1.3 or PD1.6.Sensorgrams showing the binding of the PD-1 ligands in the differentsituations are superimposed. The data shown are representative of twoseparate experiments.

(D) PD1.3 mAbs prevents PD-L1 Fc and PD-L2 Fc binding to cellsexpressing PD-1.

(E and F) The PD1.3 mAb is able to induce the IFN-γ and IL10 productionin CD4 T cells upon DC interaction. Allogenic iDC were cocultured withCD4+ T cells with anti-PD1.3, PD1.6 or isotype control. Cultures wereincubated for 5 days, supernatants were removed for cytokine analysis.The levels of IFN-γ production (E) and IL10 production (F) weredetermined in duplicate by ELISA detection. The data shown arerepresentative of two separate experiments.

FIG. 3: PD-L1 and PD-L2 do not bind to PD-1 with the same molecularmechanism.

(A) Superimposed sensorgrams representative of PD-L1 and PD-L2 bindingto PD-1 chip PD-L1 and CTLA-4 binding to CD80 chip. Proteins at 10 μg/mlwere injected for thirty seconds at a flow rate of 10 μl/min onto PD-1chip and allowed to dissociate for 120 more seconds.

(B) Superimposed sensorgrams showing short (blue) and long (red)injections of PD-L1 (up) and PD-L2 (down) onto PD-1 chip respectively.Proteins at 10 μg/ml were injected for one or seven minutes at a flowrate of 10 μl/min onto the PD-1 chip. Sensorgrams were normalized in theY axis and aligned in the X axis at the end of injection.

FIG. 4: PDL1.1 and PDL1.2 PD-L1 antibodies stabilize the binding ofPD-L1 to PD-1.

(A) Superimposed sensorgrams showing the injections of PD-L11 grecombinant proteins (grey) or the injections of PD-L11 g recombinantproteins pre-incubated with PD-L1 antibodies (black) onto PD-1 chip.PD-L1 Ig recombinant proteins at 10 μg/ml were pre-incubated withPDL1.1, PDL1.2 or PDL1.3 PD-L1 antibody Fabs at a saturatingconcentration of 100 μg/ml and injected for 10 minutes at a flow rate of10 μl/min onto the PD-1 Ig chip. Sensorgrams were normalized in the Yaxis and aligned in the X axis at the end of injection.

(B) FACS analysis on PD-L1 expressing COS cells. PDL1.1, PDL1.2 andPDL1.3 Fab mAbs were incubated with PD-L1 expressing cells for 5 or 30minutes. The binding of PD-11 g was revealed with Goat anti human (GAH)conjugated PE, the MFI ratio was indicated in the Y axis. The data shownare representative of three separate experiments.

FIG. 5: Likely mechanisms of interaction of PD-L1 with PD-1

EXAMPLE Abstract

The programmed death 1 molecule (PD-1) is involved in peripheraltolerance and in the regulation of persistent viral infections as wellas a mechanism of tumor escape from the immune system. Two ligands,PD-L1 and PD-L2 have been described that differ in tissue distribution,regulation of expression and residues involved in their binding to PD-1.We have further investigated the molecular mechanisms of PD-1interactions with its ligands using recombinant proteins and mAbs bysurface plasmon resonance and cell surface binding. We could demonstratethat both PD-L1 and PD-L2 cross-compete for PD-1 binding. Interestinglyand along the same line, one selected PD-1 mAb could interfere with thebinding of both PD-L1 and PD-L2. PD-L1 and PD-L2 bound PD-1 withcomparable affinities but striking differences standed at the level ofthe association and dissociation characteristics. Hence, PD-L1 but notPD-L2 had a delayed interaction reminiscent of a phenomenom ofconformational transition. These mechanisms were further confirmedthanks to PD-L1 mAbs that could delay the dissociation of PD-L1 fromPD-1. This mechanism was not restricted to PD-1 interaction since PD-L1behaves in a similar manner with its second ligand CD80.

Finally, CTLA-4 and PD-L1 bound to distinct, non overlapping sites onCD80. These data further emphasize the differential molecular mechanismsof interaction of both ligands to PD-1 that identify new avenues todevise mAb therapy that could prevent the binding of both ligands toPD-1 to permit an optimal blockade of immune inhibition in chronicinfection, cancer and transplantation.

Materials and Methods Constructs

Human PD-1 and CTLA-4 cDNA was generated by RT-PCR from(CD3+CD28)-activated T cells using primers shown in Table 1 andsubsequently subcloned into a DNA4 vector (Yang W C et al., Int.Immunol. 2000). The extracellular region of human PD-1 and CTLA-4 (aa 1to 152 and aa) were amplified from this plasmid using primers shown inTable 1 and cloned in frame with the Fc fragment of the human IgG1sequence using the Cos Fc Link vector (SmithKline BeechamPharmaceuticals, King of Prussia, Pa.). Human PD-L1 and PD-L2 cDNAs weregenerated by RT-PCR from brain and lung human total RNA (ClontechLaboratories, Inc) respectively using primers shown in Table 1. The samecloning protocol as PD-1 has been used to generate the full-length PD-Lsand their extracellular portions as Ig fusion proteins.

TABLE 1 Primer sequences used in this study Name Primers SequencesPD1 Full Sens ATGCAGATCCCACAGGCGCCCTGGCC (SEQ ID NO: 1) length antisenseTCAGAGGGGCCAAGAGCAGTGTCCATC (SEQ ID NO: 2) PD1 sensGCGAATTCATGCAGATCCCACAGGCGCCC (SEQ ID NO: 3) Extracellular antisenseCTTCCCGTCTTCACGGGAGCCGGCTG (SEQ ID NO: 4) PD-L1 sensATGAGGATATTTGCTGTCTTTATATTC (SEQ ID NO: 5) Full length antisenseTTACGTCTCCTCCAAATGTGT (SEQ ID NO: 6) PD-L1 sensGCGAATTCATGAGGATATTTGCTGTCTTTAT (SEQ ID NO: 7) Extracellular antisenseCCGGTACCTTCTGGGATGACCAATTCAGCTG (SEQ ID NO: 8) PD-L2 sensACGCCAAATTTTGAGTGCTT (SEQ ID NO: 9) Full length antisenseTGAAAAGTGCAAATGGCAAG (SEQ ID NO: 10) PD-L2 sensGCGAATTCATGATCTTCCTCCTGCTAATGTTG (SEQ ID NO: 11) Extracellular antisenseCCGGTACCGTCAATGCTGGCCAAAGTAAG (SEQ ID NO: 12)

PD1, PD-L, PD-L2, CTLA-4 and CD80 Soluble Human Ig Fusion Proteins

The chimeric cDNA were constructed by ligating the extra-cellular domainof PD1, PD-L1, PD-L2 and CTLA-4 with the Fc fragment of the human IgG1sequence using the Cos Fc Link vector.

Cos cells were cultured in DMEM 10% FBS with 2 mM L-Glutamine andtransfected in CHO-S-SFM II medium (from Invitrogen) without FBS withDNA plasmid construct in CFL vector with FuGENE 6 Transfection reagentaccording to the manufacturer's protocol (ROCHE). The culturesupernatant were collected seven days after transfection, filtered andloaded on a 5-ml Affigel protein A column according to themanufacturer's protocol (Bio-rad, Hercules, Calif.). After washing, theproteins were eluted with a 0.1 mol/L citrate buffer, pH 3.5,concentrated, and dialyzed against phosphate-buffer saline (PBS).Purification steps were monitored by ELISA using a sandwich revelationsystem composed with coated antibody against human IgG-UNLB and humanIgG-AP (Southern Biotechnology Associates) and revealed by pNPP substrat(Sigma). Purity and quality of the human Ig fusion proteins werecontrolled by gel electrophoresis and by cell surface staining on humanPD1, PD-L1, PD-L2 or CTLA-4 transfected COS cells line respectively.CD80 Fc was purchased from R&D.

Generation of Anti-Human PD1, PD-L1, PD-L2 and CD80 Monoclonal Antibodyand Fab Fragmentation

MAbs to human PD1, PD-L1 and PD-L2 were produced similarly. FemaleBALB/c mice were immunised by IP injection with 10 μg of human Ig fusionprotein with Freund adjuvant. Immunisation was repeated three times at 2weeks intervals, the fourth immunisation was made by IV injecting with10 μg fusion protein in the codal tail. Three days later spleen cellswere fused with X63Ag8 myeloma cells with PEG 1500 (Roche) and clonedwith HAT selection (Sigma) and Hybridoma cloning factor (HCF fromOrigen). The hybridoma supernatants were screened by cell surfacestaining human PD1, PD-L1 or PD-L2 transfected COS cells linerespectively and for lack of reactivity with untransfected COS cells.Clones PD1.3 (mouse, IgG2b) and PD1.6 (mouse, IgG1), PDL1.1, PDL1.2 andPDL1.3 (mice, IgG1) and PD-L2 (mouse, IgG1) were produced by liquidascitis production, purified with protein A affinity column and chosenas reagents for FACS and Biacore analysis and functional studies. Thetransitory transfect COS cells were obtained with FuGENE 6 Transfectionreagent according to the manufacturer's protocol (ROCHE). CD80 mAb2D10.4 as been previously reported.

Fab fragmentation was performed using papain with ImmunoPure FabPreparation Kit according to the manufacturer's protocol (PIERCE). MAbswhole and Fab were subjected to reducing SDS-PAGE and the gel wasstaining with Coomassie blue, no contaminating whole mAb was present inthe Fab preparation (data not shown). Fab retained their capacity tobind to the respectively receptors. FACS analysis with whole mAbs andtheir Fab revealed approximately the same mean fluorescence intensityupon binding to PD-1, PD-L1 or PD-L2 transfected COS cells.

FACS Analysis

Cos-7 cell line was cultured in DMEM 10% FBS with 2 mM of L-Glutamine(Invitrogen). The staining of transiently transfected COS cells followedthe basic procedure. Briefly, cells were incubated with optimizeddilution of the mAbs, were washed in cold PBS with 2% FBS and 0.02%sodium azide and incubated with goat anti mouse (GAM) conjugated withFITC (Beckman Coulter). After washes, cells were analyzed on a FACSCANTO flow cytometer (Becton Dickinson).

To test the effect of PDL1.1 and PDL1.2 anti-PD-L1 non blockingantibodies on the binding of PD-L1 to PD-1, 4μg/ml of PD-1 Ig proteinwere added on PD-L1 transfected COS cells untreated or preincubated for5 or 30 minutes with 7.5μg/ml of PDL1.1, PDL1.2 or PDL1.3 Fabs mAbs. Thebinding of PD-11 g was revealed with Goat anti human (GAH) conjugatedPE. The results are expressed as mean fluorescence intensity (MFI). TheMFI ratio was calculated by the MFI of Fab preincubated cells/MFI ofuntreated cells.

Biacore Experiments

Surface plasmon resonance measurements were performed on a Biacore 1000upgrade apparatus (Biacore GE Healthcare) at 25° C. In all Biacoreexperiments HBS-EP buffer (Biacore GE Healthcare) served as runningbuffer and sensorgrams were analyzed with Biaevaluation 4.1 software.

For protein immobilization, recombinant proteins were immobilizedcovalently to carboxyl groups in the dextran layer on a Sensor Chip CM5.The sensor chip surface was activated with EDC/NHS(N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride andN-hydroxysuccinimide (Biacore GE Healthcare)). Proteins were diluted to10 μg/ml in coupling buffer (10 mM acetate, pH 5.2) and injected untilthe appropriate immobilization level was reached (i.e. 1000 to 1200 RU).

Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

In order to determine the affinity of proteins serial dilutions from 0.3to 30 nanoM and from 1.37 to 90 nanoM of soluble antibodies andrecombinant proteins respectively were injected for 2 min at a constantflow rate of 40 μl/min on dextran layers containing immobilizedrecombinant target proteins and allowed to dissociate for 3 min beforeregeneration by a eight second injection of 500 mM NaCl and 10 mM NaOHbuffer.

The resulting sensorgrams were analysed by global fitting using theappropriate model.

For surface competitive binding inhibition experiments, the solubleanalytes were injected at a constant concentration of 10 μg/ml ondextran layers containing appropriate immobilized recombinant targetproteins. Each competition cycle consisted of three injection steps of 2min at 10 μl/min constant flow rate. Firstly, one analyte is injectedtwice. Secondly, without removing the first analyte, a second analyte isinjected and sensorgrams and RU values are monitored. Second analytesensorgram is compared to the sensorgram obtained when this analyte isinjected directly on nude recombinant target proteins. Percentage ofsecond analyte binding inhibition by first analyte (I₂₋₁) was determinedfrom RU values obtained 10 second after the end of injections, using thefollowing formula: I₂₋₁=(1−(RU₂₄/RU₂))*100. RU₂₋₁ and RU₂ are secondanalyte RU values monitored in the presence and in the absence of firstanalyte respectively. After each cycle, Sensorchips were regenerated byeight second injection of 500 mM NaCl and 10 mM NaOH buffer at flow rateof 40 μl/min.

For solution inhibition experiments, the soluble recombinant proteins ata constant concentration of 10 μg/ml were pre-incubated with increasingconcentrations of cognate recombinant ligands (from 0 to 60 μg/ml) orantibodies (from 0 to 80 μg/ml and 0 to 120 μg/ml for PD-1 mAbs, PD-L2mAbs and PD-L1 mAbs respectively) and injected for 2 minutes at a flowrate of 10 μl/min onto the appropriate chips. RU values were monitored10 seconds after the end of injection. After each cycle, Sensorchipswere regenerated by eight second injection of 500 mM NaCl and 10 mM NaOHbuffer at flow rate of 40 μl/min.

To measure the stabilization effect of PDL1.1 and PDL1.2 antibodies onPD-L1 binding to PD-1, the soluble recombinant PD-L1 Ig proteins at aconstant concentration of 10 μg/ml were pre-incubated with a saturatingconcentration of 100 μg/ml of PDL1.1 and PDL1.2 antibody Fabs andinjected for 10 minutes at a flow rate of 10 μl/min onto the PD-1 chips.The sensorgrams were monitored, normalized to 100 RU in the Y axis andcompared to those obtained without pre-incubation with anti-PD-L1antibody Fabs.

Preparation of Immature Monocyte-Derived Dcs (iDC)

iDCs were prepared from monocytes according to previously establishedprotocols (Charbonnier et al, Eur J Immunol. 1999; 29(8):2567-78) withmodifications. PBMC were obtained from healthy individual volunteers andisolated by fractionation over Lymphoprep™ Axis-Shield (ABCys) gradientcentrifugation. Monocytes were obtained from PBMC by negative selectionwith a Monocyte Isolation Kit II human according to the manufacturer'sprotocol (Miltenyi Biotec). Monocytes were cultured in 6-well plates at2.5×10⁶ cells/well (Falcon, BD Biosciences) in RPMI 1640 (Invitrogen)medium containing 10% FBS and differentiated five days with 20 ng/ml ofrecombinant human Interleukin 4 (IL-4) and 100 ng/ml of recombinanthuman GM-CSF (ABCys French society). iDcs were consistently CD14 andCD83 neg, >92% CD1a, >96% CD11b, >80% HLADR, >20% CD80 cells.

Allogenic Stimulation of CD4⁺ T Cells with iDC

CD4⁺ T cells were isolated from PBMC with negative selection with CD4⁺ TCell Isolation Kit II human according to the manufacturer's protocol(Miltenyi Biotec). CD4⁺ T cells were routinely >95% pure. CD4⁺ T cells(2×10⁵/well) were cocultured with 2×10⁴ iDC/well in triplicate in96-well flat-bottom plates (Falcon; BD Biosciences) in 200 μl of RPMI1640 (Invitrogen) supplied with 10% FBS, with mAbs to PD-1, PD-L1 andPD-L2 at various concentration. Isotype matched mAbs (B9.3, mouse IgG1and B9.4, mouse IgG2b) were used as negative controls. Cultures wereincubated for 5 days.

ELISA for Cytokine Analysis

Supernatants were then collected, IFN-γ and IL10 production weredetected in culture supernatants by ELISA detection using OptEIA™ humanIFN-γ and IL10 Set according to the manufacturer's protocol (BDBiosciences). The limit of detection was 4 pg/ml.

Results

Characterization of PD-1, PD-L1, PD-L2 mAbs and PD-11 g, PD-L1 Ig andPD-L2 Ig Fusion Proteins

In order to perform this study we have made mAbs directed against PD-1and its ligands and used them as Fabs and soluble fusion proteinscorresponding to extracellular domain of PD-1, PDL-1 and PDL-2 and theFc portion of human IgG1. We investigated the binding characteristics ofboth Fab fragments of the mAbs and fusion proteins by SPR using aBiacoreT100 and flow cytometry on transfected cells (data not shown).

By kinetic analysis using SPR Fab fragments of mAbs KD ranged from 0.26to 41 nM.

The two PD-1 mabs, PD1.3 and PD1.6 did not cross bind (data notshown).The three PD-L1 mAbs delineate two groups with PDL1.1 and PDL1.2which cross bind on one hand whereas PDL1.3 epitope is independent ofthe two other mAbs (data not shown).

We next investigated whether PD-L1 and PD-L2 could bind to PD-1 coupledto CM5 chip and reciprocally. We observed that covalent couplinginactivates the PD-L2 recombinant protein and prevents PD-1 Fc and PD-L2mAb binding (data not shown) indicating that the binding site probablycontains free NH2, whereas it had no impact on PD-L1 and PD-L2 bindingto immobilized PD-1 (FIG. 1 and FIG. 4A) nor on PD-1 binding toimmobilized PD-L1 (FIG. 1). Similar experiments where made using CD80and CTLA-4 proteins.

Taken together, these results demonstrate that the three fusion proteinsbound to their cognate receptors and Fabs fragments specifically boundto their target coupled to CM5 sensor chips.

PD-L1 and PD-L2 Cross Compete for PD-1 Binding

We next investigated whether PD-L1 and PD-L2 could bind together to PD-1or cross-competed for PD-1 occupancy.

As shown in FIG. 1, we performed a SPR analysis. The PD-1 chips werepre-incubated with increasing amount of PD-L2 (from 0 to 1000 RU ofbound PD-L2) and PD-L1 was injected without removing bound PD-L2.Sensorgrams show that PD-L1 can outcompete for PD-L2 upon increasingPD-L2 occupancy. (FIG. 1 panel A). In another experimental setting, wepre-incubated PD-1 recombinant proteins at 10 μg/ml with increasingconcentrations of PD-L2 (from 0 to 60 μg/ml) and injected the complexesonto the PD-L1 chip. PD-L2 pre-incubation prevented PD-1 binding toPD-L1 in a dose dependent manner (FIG. 1, panel B).

These data demonstrate that both ligands prevent the binding of theother PD-1 ligand and that consequently the previous binding of PD-L1 orPD-L2 to PD-1 would prevent the interaction with the other ligand in adose dependent manner. Same results were obtained by flow cytometry ontransfected cells (data not shown). Human PD-L1 interacts with humanCD80 (FIG. 1C). In order to analyze a possible interference of thebinding of PD-L1 to CD80 in presence of PD-1, we performedpre-incubation experiments and show that pre-incubation of PD-L1 withPD-1 prevents PD-L1:CD80 interaction (FIG. 1C). We next tested theinteraction of CD80 with CTLA-4 and PD-L1. As already reported, there isa strong binding of CD80 to CTLA4 that is prevented by CTLA-41 g itselfbut also the CD80 mAb 2D10.4 (FIG. 1E). Finally, we tested theinteraction of CTLA-4 and PD-L1 with CD80 (FIG. 1D). The previousbinding of CTLA-4 to CD80 does not prevent PD-L1 binding. The CD80 mAb2D10.4 did not prevent PD-L1 binding to CD80 whereas it abrogatedcompletely CTLA-4-CD80 interaction. These experiments demonstrate thatCTLA-4 and PD-L1 can both bind at the same time with human PD-L1.

PD1.3 Mab Prevents the Binding of both PD-L1 and PD-L2 to PD-1

In an additional set of experiments, we investigated whether PD-1 mAbscould in a reverse way prevent the binding of both PD-L1 and PD-L2. Asshown in FIG. 2 panels B and C, anti-PD1.3 but not anti-PD1.6 completelyinhibited the binding of both PD-L1 (FIGS. 2B and 2C upper panels) andPD-L2 (FIGS. 2B and 2C lower panels) to PD-1. In a reciprocal manner,PD1.3 mAb blocked in a dose dependent way the binding of PD-1 Fc toPD-L1 chips (data not shown). By FACS analysis, PD1.3 mAb can inhibitPD-1 Fc binding to PD-L1 and PD-L2 expressing cells (FIG. 2C).

PD-1 Mabs can Modulate T Cell Activation by Allogeneic ImmatureDendritic Cells

We next investigated the functional capabilities of the mAbs directedagainst PD-1. We tested their ability to induce the activationallogeneic T cells against a suboptimal activation namely immaturemonocytes derived dendritic cells. The mAb inhibiting PD-1 ligandinteraction, PD1.3 but not the non inhibitory PD1.6 was able to enhancethe activation of CD4 T cells as indicated by an increased INFγ and IL10production (FIG. 2 panels D) and T cell proliferation (data not shown).

Altogether, these data demonstrate that the two ligands compete for PD-1binding and conversely that an inhibitory anti-PD-1 mAb can readilyprevent PD-1 ligands binding and as consequence enhance T cellactivation.

PD-L1 and PD-L2 Differ in their Molecular Mechanisms of PD-1 Binding

We next investigated the mechanisms of PD-L1 and PD-L2 binding to PD-1.To pinpoint the different steps of this interaction we performed acareful kinetic study. Kinetic binding assays were performed todetermine the equilibrium dissociation constant between PD-1 and PD-L1and PD-L2 fusion proteins. The binding data were first analyzed usingthe 1:1 Langmuir model. For PD-L2/PD-1 the fitting was very good andyielded a KD of 9.97 nM. Though both recombinant analytes were bivalentthe Langmuir model fitted very well to the data indicating that the twobinding sites of each molecule probably interact at the same time. Thus,the KD value most likely represents an avidity value.

For PD-L1/PD-1, as well as PD-L1/CD80 fitting using the Langmuir modelwas very bad and inappropriate.

Indeed, as shown in FIG. 3 panel A, the two ligands interacted with PD-1with distinct features. PD-L1 associated rapidly and dissociated alsovery fast. In contrast PD-L2 binding was delayed with a different slopeand more robust. The residual values strongly differed. Hence, twodistinct phenomenons were observed in the PDL-L1 PD-1 dissociationphase. An early phase, during which PD-L1 rapidly dissociated from PD-1at the beginning of the dissociation, was followed by a latter phasecharacterized by a low dissociation rate with signals appearing morestable, whereas PD-L2 PD-1 dissociation was more homogeneous. From thedifferent models tested for fitting, the “conformational modificationmodel” gave the best and reliable fit and yielded an apparentdissociation constant (KD#) of 10.7 nM and 56.5 nM on PD-1 chips.

It can be predicted from the “conformational modification model” thatincreasing the time of injection would also increase the stability ofbinding. To test this hypothesis we then compared the binding of PD-L1and PD-L2 to PD-1 chips following two settings, 10 μg/ml PD-L1 or PD-L2fusion proteins were injected during 7 or 1 minute respectively. Thesensorgrams depicted in FIG. 3B clearly demonstrate that the contacttime influence the stability of PD-L1 binding to the PD-1 chip. Thedissociation of PD-L1 to the PD-1 chip was slower when the injectiontime was longer.

As a control, for PD-L2 the dissociation kinetic was not affected byincreasing the injection time (FIG. 3B).

Altogether, these data demonstrate striking differences in themechanisms of interaction of PD-L1 and PD-L2 with PD-1. The dataobtained with PD-L1 fit with a model where a conformation change will beneeded for its efficient binding with its receptor.

We finally analyzed whether this conformational change model wasrestricted to PD-L1:PD-1 interaction and hence due to the ligand orobserved with another PD-L1 ligand like CD80. As shown in FIG. 3A, PD-L1binding to CD80 was also associated with the conformational changemodel. Altogether, these data indicate that PD-L1 and PD-L2 differ intheir binding to PD-1. Whereas bind to its ligands, namely PD-1 and CD80with similar association and dissociation mechanisms.

Non Blocking PD-L1 Mabs Increase the Binding of PD-L1 to PD-1

We have also tested the ability of PD-L1 and PD-L2 mabs to prevent theinteraction of PD-L1 and PD-L2 to PD-1. Anti-PD-L2 and anti-PDL1.3 mAbsprevented PD-1/PD-L2 and PD-L1 interaction respectively (data not shownand). However, anti-PDL1.1 and anti-PDL1.2 did not inhibit PD-L1/PD-1interaction (data not shown). As PD-L1 binding to PD-1 could induce aconformational modification of PD-L1 we reasoned that these two mAbsthat do not interfere with PD-1/PD-L1 binding might affect other PD-L1regions that might be critical for ligand -receptor interaction. Hence,we also investigated whether the anti-PD-L1 antibodies that do not blockbinding to PD-1 could influence this phenomenon. To do so, PD-L11 gproteins were incubated with a saturating concentration of PDL1.1 andPDL1.2 antibody Fabs and injected onto the PD-1 chip (FIG. 4 A). Weobserved that pre-incubation with both non blocking anti-PD-L1antibodies clearly modifies the dissociation of PD-L11 g proteins fromPD-1 chip. The PD-L1 dissociation is slower when PD-L1 is bound toeither PDL1.1 or PDL1.2 Fabs. The binding of PDL1.1 or PDL1.2 Fabs toPD-L1 seems to increase the stability of the PD-L1 PD-1 interaction, maybe by influencing the conformational modification of PD-L1. These dataseem in line with the “conformational modification model” that wepreferred to choose for the fitting of PD-L1 binding to PD-1. However,the fact that the conformational transition phenomenon was observableusing the Biacore indicates that the modification of PD-L1 state is nota fast phenomenon. Taking in account these considerations we nextinvestigated whether the conformational modification could be observedon native PD-L1 molecules expressed at the cell surface.

Thus, the stabilizing effect of PDL1.1 and PDL1.2 non blockingantibodies on the binding of PD-L1 to PD-1 was analysed at a cellularlevel. PD-L1 expressing COS cells were incubated for 5 or 30 minuteswith the three different anti-PD-L1 Fabs. Then, PD-11 g protein bindingwas tested by FACS analysis. The PDL1.1 Fab pre-incubation induced anincrease of the MFI due to PD-1 protein binding (FIG. 4B). This increaseoccurred as early as 5 minutes post-Fab injection and was furtherenhanced after 30 minutes. This result was in accordance with the dataof the SPR analysis (FIG. 4A). As shown in FIG. 4B, both non blockingPDL1.1 and PDL1.2 induce an increase of PD-1 binding. In control, theblocking PDL1.3 Fab impaired this PD-1 binding (FIG. 4B). Same resultswere obtained using immature DC expressing PD-L1 (data not shown). Takentogether, the non blocking PD-L1 mAbs are not neutral but in factpromote the binding of PD-L1 to PD-1.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A PD-L1 antibody, which stabilizes the binding of PD-L1 to PD-1.
 2. APD-L1 antibody obtainable from the hybridoma accessible under CNCMdeposit number I-4080 or I-4081.
 3. A PD-L1 antibody, which stabilizesthe binding of PD-L1 to PD-1, comprising the CDRs of the PD-L1 antibodyof claim
 2. 4. A method for treating the human or animal body bytherapy, comprising administering a PD-L1 antibody according to any oneof claims 1 to 3 to said human or animal body.
 5. A method according toclaim 4 for treating an autoimmune disease, transplantation rejection ora graft versus host disease.
 6. A hybridoma cell line selected from thegroup consisting of CNCM I-4122, CNCM I-4080 and CNCM I-4081.
 7. A PD-1antibody obtainable from the hybridoma accessible under CNCM depositnumber I-4122.
 8. A PD-1 antibody comprising the CDRs of the PD-1antibody of claim
 7. 9. A method for treating the human or animal bodyby therapy, comprising administering a PD-1 antibody according to claim7 to said human or animal body.
 10. A method according to claim 9 forwherein said therapy is for a cancer or a chronic infection.
 11. Avaccine for the treatment of a cancer or a chronic infection comprisinga PD-1 antibody according to claim
 7. 12. A kit for the treatment of acancer or a chronic infection comprising: a) a PD-1 antibody accordingto claim 7; and b) a vaccine for the treatment of a cancer or a chronicinfection.
 13. A method for treating the human or animal body bytherapy, comprising administering a PD-1 antibody according to claim 8to said human or animal body.
 14. A method according to claim 13 whereinsaid therapy is for cancer or a chronic infection.
 15. A vaccine for thetreatment of a cancer or a chronic infection comprising a PD-1 antibodyaccording to claim
 8. 16. A kit for the treatment of a cancer or achronic infection comprising: a) a PD-1 antibody according to claim 8;and b) a vaccine for the treatment of a cancer or a chronic infection.